Identification of circulatory microRNA based biomarkers for early pregnancy diagnosis in buffalo.

Introduction: The most crucial factor in improving animal reproduction efficiency is early pregnancy diagnosis. Early diagnosis not only reduces the time interval between two calvings but also aids farmers in identifying open animals, thereby preventing significant milk production losses. Therefore, the objective of this study was to discover circulatory miRNAs that would be useful for early pregnancy diagnosis in buffalo. Material and methods: Blood samples were taken on 0, 6th, 12th, and 18th day after artificial insemination from pregnant animals (n = 30) and non-pregnant animals (n = 20). During these stages of pregnancy, total RNA was extracted, and a small RNA library was subsequently generated and sequenced on the Illumina platform. Subsequently, Real-time PCR was used to validate the findings. Results and discussion: There were 4,022 miRNAs found during the pregnancy, with 15 of those lacking sequences and 4,007 having sequences already in the database. From the beginning of pregnancy until the 18th day, 25 of these miRNAs showed a substantial shift in expression levels in the maternal blood, with a change more than two logs. Furthermore, based on qPCR results, 19 miRNAs were found to be more abundant in pregnant animals than in non-pregnant animals. We used target prediction analysis to learn how maternally expressed miRNAs relate to fetal-maternal communication. In conclusion, miRNA based biomarkers that could be associated with the diagnosis of pregnancy were identified including miR-181a and miR-486 highly upregulated on the 18th day of pregnancy. This study also provides a comprehensive profile of the entire miRNA population in maternal buffalo blood during the early stages of pregnancy.

Development of a multiplexed Luminex assay for simultaneous detection of enteric viruses in cattle.

Viral and bacterial gastroenteritis and diarrhea have long been a problem in livestock with devastating effects on animal health and production causing a heavy financial burden on producers. Therefore, the bead-based multiplex detection assay was created for simultaneous detection of three livestock viral diarrheic agents viz. bovine rotavirus (BRV), bovine coronavirus (BCoV) and bluetongue virus (BTV). The primers and probes for triplex MAGPIX assay for simultaneous detection of three enteric viruses were designed and the assay was optimized for hybridization temperature, primer-probe and bead concentrations. The newly developed MAGPIX assay was used to determine the prevalence of these diarrhea-associated viruses by testing 200 fecal samples collected from Haryana state of India during 2018-2019. The limit of detection of the developed triplex assay was 1 × 105, 1 × 104, and 1 × 105 RNA copies for BRV, BCoV, and BTV, respectively, being lower than the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). However, it was higher than the conventional RT-PCR, showing it to be more sensitive. The newly developed MAGPIX assay was a rapid, cost-effective and high throughput diagnostic tool for identification of three major entero-pathogenic diarrhea associated viruses, either alone or in tandem, with the aim to prevent and control viral diarrhea in animals.

Rapid detection of porcine sapelovirus by reverse transcription recombinase polymerase amplification assay.

BACKGROUND: Porcine Sapelovirus (PSV) infection has been confirmed in pigs worldwide, mostly asymptomatic, but in some cases, it can lead to significant issues in the gastrointestinal, respiratory, neurological, or reproductive systems. PSV is considered an emerging pathogen of porcine species. Recombinase polymerase amplification (RPA) is a simple and fast isothermal technique that uses three enzymes for amplification without the use of any sophisticated equipment. METHODS AND RESULTS: The reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and optimized for field based detection of PSV. The assay was developed by targeting 5´UTR region of PSV genome and optimized for reaction time, temperature, primer and MgOAc concentration. The analytical sensitivity and specificity of assay was determined. The assay was evaluated on 85 porcine faecal samples collected from field. In addition to conventional format, this assay was also optimized for visual dye-based detection format and lateral flow strips based detection (in combination with probe). The developed assay works at constant temperature of 35 °C for 20 min with forward primer concentration 20pm, reverse primer concentration 10pm and MgOAc concentration of 14mM. This assay is highly sensitive and detects up to 28 copies of viral nucleic acid both in the conventional as well as in fluorescent dye based detection format. Using the newly developed assay 21 samples out of 85 samples were found positive, showing positivity rate of 24.7%. The positivity rate of RT-RPA assay corroborated with the gold standard RT-PCR test. CONCLUSIONS: This study presented the development of an RT-RPA isothermal assay for rapid and accurate detection of PSV. The assay is highly sensitive, specific, works at a low and constant temperature, does not require any high-end instrument and can be a potential diagnostics tool for pen-side testing of PSV in the field conditions. The newly developed RT-RPA assay could successfully detect PSV circulating in swine population of Haryana, India. This is a first report of this kind from the region.

Development of real-time RT-PCR systems for detection and quantitation of bovine enteric viral pathogens.

The enteric viruses in animals are responsible for severe and devastating losses to the livestock owners with a profound negative impact on animal, health, welfare, and productivity. These viruses are usually transmitted via the feco-oral route and primarily infect the digestive tract of the humans, bovines and different mammals as well as birds. Some of the important enteric viruses in ruminants are: Rotavirus A (RVA), Peste des petits virus (PPRV), Norovirus (NV), Bovine corona virus (BoCV) and Bluetongue virus (BTV). In the present study, sensitive, specific and reliable TaqMan probe-based RT-qPCRs were developed and standardized for the rapid detection and quantification of enteric viruses from fecal samples. The assays result in efficient amplification of the RVA, BTV and BoCV RNA with a limit of detection (LoD) of 5, 5 and 4 copies, respectively, which is 1000 times more sensitive than the traditional gel-based RT-PCR. The reproducibility of each assay was satisfactory, thus allowing for a sensitive and accurate measurement of the viral RNA load in clinical samples. In conclusion, real time PCR developed for these viruses are highly specific and sensitive technique for the detection of diarrheic viral pathogens of cattle and buffalo.